The proposed investigation will be a continuation of earlier studies and will examine various aspects of the role of nuclear genes in the post transcriptional control of ATPase biogenesis. Yeast nuclear ATPase mutants which have been found to complement a well characterized assembly defective mutant will be examined using genetic immunological and biochemical methods to further define the assembly pathway of the enzyme. More direct analysis of the post translational events of ATPase assembly will be provided using precursor forms of the ATPase complex which are made in the in vitro translations or which accumulate in vivo when the oxidative energy source is limiting. These precursor subunits of the F1 ATPase will be used as substrates for the assay, characterization and purification of the membrane bound processing enzyme which is involved in a post translational processing event. The biological significance of F1 ATPase subunits which are present but unassembled in wild type mitochondrial membrane will be determined. Radiochemical pulse and pulse chase labeling experiments will be performed to deterine if these unassembled subunits are transient components or if they label with the same kinetics as the assembled enzyme. Additional insight into the mechanism of post transcriptional control by a single nuclear mutation will be obtained by isolation and characterization of the specific mRNA(s) for the F1 ATPase. RNA will be extracted from Agarose-Urea gels and identified by in vitro translation and immunoprecipitation techniques with subunit specific antisera. Analysis of purified mRNA will immediately distinguish the organization of F1 ATPase coding sequences on either a single high molecular weight RNA transcript or a multiple subunit specific transcripts. These studies will provide the tools required for a more detailed analysis of membrane assembly at the level of the gene.